34 results on '"Münzer P"'
Search Results
2. Pivotal role of PDK1 in megakaryocyte cytoskeletal dynamics and polarization during platelet biogenesis.
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Geue S, Aurbach K, Manke MC, Manukjan G, Münzer P, Stegner D, Brähler C, Walker-Allgaier B, Märklin M, Borst CE, Quintanilla-Fend L, Rath D, Geisler T, Salih HR, Seizer P, Lang F, Nieswandt B, Gawaz M, Schulze H, Pleines I, and Borst O
- Subjects
- Animals, Blood Platelets cytology, Humans, Megakaryocytes cytology, Mice, Mice, Knockout, 3-Phosphoinositide-Dependent Protein Kinases metabolism, Blood Platelets metabolism, Cytoskeleton metabolism, Megakaryocytes metabolism, Thrombopoiesis physiology
- Abstract
During thrombopoiesis, megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Phosphoinositide-dependent protein kinase-1 (PDK1) is required for Ca2+-dependent platelet activation, but its role in MK development and regulation of platelet production remained elusive. The present study explored the role of PDK1 in the regulation of MK maturation and polarization during thrombopoiesis using a MK/platelet-specific knockout approach. Pdk1-deficient mice (Pdk1-/-) developed a significant macrothrombocytopenia as compared with wild-type mice (Pdk1fl/fl). Pdk1 deficiency further dramatically increased the number of MKs without sinusoidal contact within the BM hematopoietic compartment, resulting in a pronounced MK hyperplasia and a significantly increased extramedullary thrombopoiesis. Cultured Pdk1-/- BM-MKs showed impaired spreading on collagen, associated with an altered actin cytoskeleton structure with less filamentous actin (F-actin) and diminished podosome formation, whereas the tubulin cytoskeleton remained unaffected. This phenotype was associated with abrogated phosphorylation of p21-activated kinase (PAK) as well as its substrates LIM domain kinase and cofilin, supporting the hypothesis that the defective F-actin assembly results from increased cofilin activity in Pdk1-deficient MKs. Pdk1-/- BM-MKs developed increased ploidy and exhibited an abnormal ultrastructure with disrupted demarcation membrane system (DMS). Strikingly, Pdk1-/- BM-MKs displayed a pronounced defect in DMS polarization and produced significantly less proplatelets, indicating that PDK1 is critically required for proplatelet formation. In human MKs, genetic PDK1 knockdown resulted in increased maturity but reduced platelet-like particles formation. The present observations reveal a pivotal role of PDK1 in the regulation of MK cytoskeletal dynamics and polarization, proplatelet formation, and thrombopoiesis., (© 2019 by The American Society of Hematology.)
- Published
- 2019
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3. Functional Relevance of the Anaphylatoxin Receptor C3aR for Platelet Function and Arterial Thrombus Formation Marks an Intersection Point Between Innate Immunity and Thrombosis.
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Sauter RJ, Sauter M, Reis ES, Emschermann FN, Nording H, Ebenhöch S, Kraft P, Münzer P, Mauler M, Rheinlaender J, Madlung J, Edlich F, Schäffer TE, Meuth SG, Duerschmied D, Geisler T, Borst O, Gawaz M, Kleinschnitz C, Lambris JD, and Langer HF
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- Animals, Blood Platelets immunology, Calcium Signaling, Complement Activation, Complement C3 genetics, Complement C3 immunology, Complement C3 metabolism, Disease Models, Animal, Humans, Mice, Inbred C57BL, Mice, Knockout, Myocardial Infarction immunology, Platelet Activation, Platelet Glycoprotein GPIIb-IIIa Complex metabolism, Platelet Glycoprotein GPIb-IX Complex genetics, Platelet Glycoprotein GPIb-IX Complex metabolism, Receptors, Complement deficiency, Receptors, Complement genetics, Receptors, Complement immunology, Stroke immunology, Thrombosis immunology, Blood Coagulation, Blood Platelets metabolism, Immunity, Innate, Myocardial Infarction blood, Receptors, Complement blood, Stroke blood, Thrombosis blood
- Abstract
Background: Platelets have distinct roles in the vascular system in that they are the major mediator of thrombosis, critical for restoration of tissue integrity, and players in vascular inflammatory conditions. In close spatiotemporal proximity, the complement system acts as the first line of defense against invading microorganisms and is a key mediator of inflammation. Whereas the fluid phase cross-talk between the complement and coagulation systems is well appreciated, the understanding of the pathophysiological implications of such interactions is still scant., Methods: We analyzed coexpression of the anaphylatoxin receptor C3aR with activated glycoprotein IIb/IIIa on platelets of 501 patients with coronary artery disease using flow cytometry; detected C3aR expression in human or murine specimen by polymerase chain reaction, immunofluorescence, Western blotting, or flow cytometry; and examined the importance of platelet C3aR by various in vitro platelet function tests, in vivo bleeding time, and intravital microscopy. The pathophysiological relevance of C3aR was scrutinized with the use of disease models of myocardial infarction and stroke. To approach underlying molecular mechanisms, we identified the platelet small GTPase Rap1b using nanoscale liquid chromatography coupled to tandem mass spectrometry., Results: We found a strong positive correlation of platelet complement C3aR expression with activated glycoprotein IIb/IIIa in patients with coronary artery disease and coexpression of C3aR with glycoprotein IIb/IIIa in thrombi obtained from patients with myocardial infarction. Our results demonstrate that the C3a/C3aR axis on platelets regulates distinct steps of thrombus formation such as platelet adhesion, spreading, and Ca
2+ influx. Using C3aR-/- mice or C3-/- mice with reinjection of C3a, we uncovered that the complement activation fragment C3a regulates bleeding time after tail injury and thrombosis. Notably, C3aR-/- mice were less prone to experimental stroke and myocardial infarction. Furthermore, reconstitution of C3aR-/- mice with C3aR+/+ platelets and platelet depletion experiments demonstrated that the observed effects on thrombosis, myocardial infarction, and stroke were specifically caused by platelet C3aR. Mechanistically, C3aR-mediated signaling regulates the activation of Rap1b and thereby bleeding arrest after injury and in vivo thrombus formation., Conclusions: Overall, our findings uncover a novel function of the anaphylatoxin C3a for platelet function and thrombus formation, highlighting a detrimental role of imbalanced complement activation in cardiovascular diseases.- Published
- 2018
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4. Identification of key lipids critical for platelet activation by comprehensive analysis of the platelet lipidome.
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Peng B, Geue S, Coman C, Münzer P, Kopczynski D, Has C, Hoffmann N, Manke MC, Lang F, Sickmann A, Gawaz M, Borst O, and Ahrends R
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- Animals, Humans, Mice, Mice, Inbred C57BL, Mice, Knockout, Phosphorylcholine metabolism, Platelet Activation, Sphingosine metabolism, Thrombosis metabolism, Blood Platelets metabolism, Lipids analysis, Phosphorylcholine analogs & derivatives, Sphingomyelin Phosphodiesterase physiology, Sphingosine analogs & derivatives, Thrombosis physiopathology
- Abstract
Platelet integrity and function critically depend on lipid composition. However, the lipid inventory in platelets was hitherto not quantified. Here, we examined the lipidome of murine platelets using lipid-category tailored protocols on a quantitative lipidomics platform. We could show that the platelet lipidome comprises almost 400 lipid species and covers a concentration range of 7 orders of magnitude. A systematic comparison of the lipidomics network in resting and activated murine platelets, validated in human platelets, revealed that <20% of the platelet lipidome is changed upon activation, involving mainly lipids containing arachidonic acid. Sphingomyelin phosphodiesterase-1 (Smpd1) deficiency resulted in a very specific modulation of the platelet lipidome with an order of magnitude upregulation of lysosphingomyelin (SPC), and subsequent modification of platelet activation and thrombus formation. In conclusion, this first comprehensive quantitative lipidomic analysis of platelets sheds light on novel mechanisms important for platelet function, and has therefore the potential to open novel diagnostic and therapeutic opportunities., (© 2018 by The American Society of Hematology.)
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- 2018
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5. PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway.
- Author
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Manne BK, Münzer P, Badolia R, Walker-Allgaier B, Campbell RA, Middleton E, Weyrich AS, Kunapuli SP, Borst O, and Rondina MT
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- 3-Phosphoinositide-Dependent Protein Kinases antagonists & inhibitors, 3-Phosphoinositide-Dependent Protein Kinases blood, 3-Phosphoinositide-Dependent Protein Kinases deficiency, 3-Phosphoinositide-Dependent Protein Kinases genetics, Animals, Blood Platelets drug effects, Disease Models, Animal, Humans, Mice, Knockout, Phosphorylation, Platelet Aggregation Inhibitors pharmacology, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Mas, Pulmonary Embolism blood, Pulmonary Embolism genetics, Pulmonary Embolism prevention & control, Pyrimidines pharmacology, Signal Transduction, Thiophenes pharmacology, Thrombosis blood, Thrombosis genetics, Thrombosis prevention & control, 3-Phosphoinositide-Dependent Protein Kinases metabolism, Blood Platelets enzymology, Mitogen-Activated Protein Kinases blood, Platelet Aggregation drug effects, Proto-Oncogene Proteins c-raf blood, Pulmonary Embolism enzymology, Thrombosis enzymology, Thromboxanes blood
- Abstract
Essentials Phosphoinositide 3-kinase and MAPK pathways crosstalk via PDK1. PDK1 is required for adenosine diphosphate-induced platelet activation and thromboxane generation. PDK1 regulates RAF proto-oncogene Ser/Thr kinase (Raf1) activation in the MAPK pathway. Genetic ablation of PDK1 protects against platelet-dependent thrombosis in vivo., Summary: Background Platelets are dynamic effector cells with functions that span hemostatic, thrombotic and inflammatory continua. Phosphoinositide-dependent protein kinase 1 (PDK1) regulates protease-activated receptor 4-induced platelet activation and thrombus formation through glycogen synthase kinase3β. However, whether PDK1 also signals through the ADP receptor and its functional importance in vivo remain unknown. Objective To establish the mechanism of PDK1 in ADP-induced platelet activation and thrombosis. Methods We assessed the role of PDK1 on 2MeSADP-induced platelet activation by measuring aggregation, thromboxane generation and phosphorylation events in the presence of BX-795, which inhibits PDK1, or by using platelet-specific PDK1 knockout mice and performing western blot analysis. PDK1 function in thrombus formation was assessed with an in vivo pulmonary embolism model. Results PDK1 inhibition with BX-795 reduced 2-methylthio-ADP (2MeSADP)-induced aggregation of human and murine platelets by abolishing thromboxane generation. Similar results were observed in pdk1
-/- mice. PDK1 was also necessary for the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2, and cytosolic phospholipase A2, indicating that PDK1 regulates an upstream kinase in the mitogen-activated protein kinase (MAPK) pathway. We next determined that this upstream kinase is Raf-1, a serine/threonine kinase that is necessary for the phosphorylation of MEK1/2, as pharmacological inhibition and genetic ablation of PDK1 were sufficient to prevent Raf1 phosphorylation. Furthermore, in vivo inhibition or genetic ablation of PDK1 protected mice from collagen/epinephrine-induced pulmonary embolism. Conclusion PDK1 governs thromboxane generation and thrombosis in platelets that are stimulated with 2MeSADP by regulating activation of the MAPK pathway., (© 2018 International Society on Thrombosis and Haemostasis.)- Published
- 2018
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6. Ceramidase critically affects GPVI-dependent platelet activation and thrombus formation.
- Author
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Münzer P, Mittelstädt S, Geue S, Manke MC, Walker-Allgaier B, Lang F, Gawaz M, and Borst O
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- Animals, Cells, Cultured, Female, Male, Mice, Blood Platelets enzymology, Ceramidases metabolism, Platelet Aggregation, Platelet Membrane Glycoproteins metabolism, Thrombosis enzymology, Thrombosis metabolism
- Abstract
Platelet aggregation, dense granule secretion and thrombus formation are dependent on sphingolipids like ceramide and sphingosine as well as sphingosine-1 phosphate. Sphingosine/ceramide metabolism involves ceramide synthases and ceramidases. However, the role of ceramide synthase and ceramidase in the regulation of platelet function remained ill-defined. The present study determined transmission light aggregometry, employed luciferase based ATP release measurements and studied in vitro thrombus formation under high arterial shear rates in order to define the impact of pharmacological inhibition of serine palmitoyltransferase, ceramide synthase and ceramidase on platelet function. As a result, inhibition of ceramidase significantly blunted collagen related peptide (CRP) induced glyocoprotein VI (GPVI)-dependent platelet aggregation, ATP release and thrombus formation on a collagen-coated surface under shear rates of 1700
-sec . Defective platelet aggregation after ceramidase inhibition could partially be overcome by exogenous sphingosine treatment reflecting a pivotal role of ceramidase-derived sphingosine in platelet function. Inhibition of serine palmitoyltransferase and ceramide synthase did not significantly modify GPVI-dependent platelet activation. In conclusion, the present study unraveled ceramidase as a crucial player in sphingosine-induced platelet activation following GPVI-dependent signaling., (Copyright © 2018 Elsevier Inc. All rights reserved.)- Published
- 2018
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7. Extracellular Cyclophilin A Augments Platelet-Dependent Thrombosis and Thromboinflammation.
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von Ungern-Sternberg SNI, Vogel S, Walker-Allgaier B, Geue S, Maurer A, Wild AM, Münzer P, Chatterjee M, Heinzmann D, Kremmer E, Borst O, Loughran P, Zernecke A, Neal MD, Billiar TR, Gawaz M, and Seizer P
- Subjects
- Animals, Anti-Inflammatory Agents pharmacology, Antibodies, Monoclonal pharmacology, Antibodies, Neutralizing pharmacology, Basigin metabolism, Blood Platelets drug effects, Cell Adhesion, Cell Movement, Cells, Cultured, Cyclophilin A antagonists & inhibitors, Disease Models, Animal, Fibrinolytic Agents pharmacology, Humans, Inflammation prevention & control, Macrophages metabolism, Mice, Inbred C57BL, Monocytes metabolism, Peritonitis chemically induced, Peritonitis prevention & control, Protein Interaction Domains and Motifs, Rats, Thrombosis prevention & control, Blood Coagulation drug effects, Blood Platelets metabolism, Cyclophilin A blood, Inflammation blood, Peritonitis blood, Platelet Activation drug effects, Thrombosis blood
- Abstract
Cyclophilin A (CyPA) is involved in the pathophysiology of several inflammatory and cardiovascular diseases. To our knowledge, there is no specific inhibitor targeting extracellular CyPA without affecting other extracellular cyclophilins or intracellular CyPA functions. In this study, we developed an antibody-based inhibitor of extracellular CyPA and analysed its effects in vitro and in vivo . To generate a specific antibody, mice and rats were immunized with a peptide containing the extracellular matrix metalloproteinase inducer binding site and various antibody clones were selected and purified. At first, antibodies were tested for their binding capacity to recombinant CyPA and their functional activity. The clone 8H7-mAb was chosen for further experiments. 8H7-mAb reduced the CyPA-induced migration of inflammatory cells in vitro and in vivo . Furthermore, 8H7-mAb revealed strong antithrombotic effects by inhibiting CyPA-dependent activation of platelets and thrombus formation in vitro and in vivo . Surprisingly, 8H7-mAb did not influence in vivo tail bleeding time or in vitro whole blood coagulation parameters. Our study provides first evidence that antibody-based inhibition of extracellular CyPA inhibits thrombosis and thromboinflammation without affecting blood homeostasis. Thus, 8H7-mAb may be a promising compound for thrombi modulation in inflammatory diseases to prevent organ dysfunction., Competing Interests: Disclosure: None., (Schattauer GmbH Stuttgart.)
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- 2017
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8. Doxepin inhibits GPVI-dependent platelet Ca 2+ signaling and collagen-dependent thrombus formation.
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Geue S, Walker-Allgaier B, Eißler D, Tegtmeyer R, Schaub M, Lang F, Gawaz M, Borst O, and Münzer P
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- Animals, Blood Platelets cytology, Blood Platelets metabolism, Calcium metabolism, Carrier Proteins antagonists & inhibitors, Carrier Proteins metabolism, Cell Degranulation drug effects, Cells, Cultured, Female, Gene Expression Regulation, Inositol 1,4,5-Trisphosphate metabolism, Ion Transport drug effects, Male, Mice, Mice, Inbred C57BL, Peptides antagonists & inhibitors, Peptides metabolism, Phospholipase C gamma genetics, Phospholipase C gamma metabolism, Platelet Activation drug effects, Platelet Adhesiveness drug effects, Platelet Aggregation drug effects, Platelet Glycoprotein GPIIb-IIIa Complex genetics, Platelet Glycoprotein GPIIb-IIIa Complex metabolism, Platelet Membrane Glycoproteins genetics, Platelet Membrane Glycoproteins metabolism, Stress, Mechanical, Thrombosis prevention & control, Antidepressive Agents, Tricyclic pharmacology, Blood Platelets drug effects, Calcium Signaling drug effects, Carrier Proteins genetics, Doxepin pharmacology, Peptides genetics, Platelet Aggregation Inhibitors pharmacology, Platelet Membrane Glycoproteins antagonists & inhibitors
- Abstract
Platelet adhesion, activation, and aggregation are essential for primary hemostasis, but are also critically involved in the development of acute arterial thrombotic occlusion. Stimulation of the collagen receptor glycoprotein VI (GPVI) leads to phospholipase Cγ2-dependent inositol triphosphate (IP
3 ) production with subsequent platelet activation, due to increased intracellular Ca2+ concentration ([Ca2+ ]i ). Although tricyclic antidepressants have been shown to potentially impair platelet activation, nothing is hitherto known about potential effects of the tricyclic antidepressant doxepin on platelet Ca2+ signaling and thrombus formation. As shown in the present study, doxepin significantly diminished the stimulatory effect of GPVI agonist collagen-related peptide (CRP) on intracellular Ca2+ release as well as subsequent extracellular Ca2+ influx. Doxepin was partially effective by impairment of CRP-dependent IP3 production. Moreover, doxepin abrogated CRP-induced platelet degranulation and integrin αIIb β3 activation and aggregation. Finally, doxepin markedly blunted in vitro platelet adhesion to collagen and thrombus formation under high arterial shear rates (1,700-s ). In conclusion, doxepin is a powerful inhibitor of GPVI-dependent platelet Ca2+ signaling, platelet activation, and thrombus formation., (Copyright © 2017 the American Physiological Society.)- Published
- 2017
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9. PDK1 Determines Collagen-Dependent Platelet Ca2+ Signaling and Is Critical to Development of Ischemic Stroke In Vivo.
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Münzer P, Walker-Allgaier B, Geue S, Geuss E, Hron G, Rath D, Eißler D, Winter S, Schaeffeler E, Meinert M, Schaller M, Greinacher A, Schwab M, Geisler T, Kleinschnitz C, Lang F, Gawaz M, and Borst O
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- 3-Phosphoinositide-Dependent Protein Kinases deficiency, 3-Phosphoinositide-Dependent Protein Kinases genetics, Animals, Disease Models, Animal, Genetic Predisposition to Disease, Infarction, Middle Cerebral Artery blood, Infarction, Middle Cerebral Artery pathology, Inositol 1,4,5-Trisphosphate blood, Mice, Knockout, Neuropeptides blood, Phenotype, Phospholipase C gamma blood, Platelet Adhesiveness, Platelet Aggregation, Platelet Membrane Glycoproteins deficiency, Platelet Membrane Glycoproteins genetics, Thrombosis blood, Thrombosis pathology, Time Factors, rac1 GTP-Binding Protein blood, 3-Phosphoinositide-Dependent Protein Kinases metabolism, Blood Platelets enzymology, Calcium Signaling, Collagen metabolism, Infarction, Middle Cerebral Artery enzymology, Platelet Activation, Thrombosis enzymology
- Abstract
Objective: Activation of platelets by subendothelial collagen results in an increase of cytosolic Ca(2+) concentration ([Ca(2+)]i) and is followed by platelet activation and thrombus formation that may lead to vascular occlusion. The present study determined the role of phosphoinositide-dependent protein kinase 1 (PDK1) in collagen-dependent platelet Ca(2+) signaling and ischemic stroke in vivo., Approach and Results: Platelet activation with collagen receptor glycoprotein VI agonists collagen-related peptide or convulxin resulted in a significant increase in PDK1 activity independent of second-wave signaling. PDK1 deficiency was associated with reduced platelet phospholipase Cγ2-dependent inositol-1,4,5-trisphosphate production and intracellular [Ca(2+)]i in response to stimulation with collagen-related peptide or convulxin. The defective increase of [Ca(2+)]i resulted in a substantial defect in activation-dependent platelet secretion and aggregation on collagen-related peptide stimulation. Furthermore, Rac1 activation and spreading, adhesion to collagen, and thrombus formation under high arterial shear rates were significantly diminished in PDK1-deficient platelets. Mice with PDK1-deficient platelets were protected against arterial thrombotic occlusion after FeCl3-induced mesenteric arterioles injury and ischemic stroke in vivo. These mice had significantly reduced brain infarct volumes, with a significantly increased survival of 7 days after transient middle cerebral artery occlusion without increase of intracerebral hemorrhage. Tail bleeding time was prolonged in pdk1(-/-) mice, reflecting an important role of PDK1 in primary hemostasis., Conclusions: PDK1 is required for Ca(2+)-dependent platelet activation on stimulation of collagen receptor glycoprotein VI, arterial thrombotic occlusion, and ischemic stroke in vivo., (© 2016 American Heart Association, Inc.)
- Published
- 2016
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10. CD44 sensitivity of platelet activation, membrane scrambling and adhesion under high arterial shear rates.
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Liu G, Liu G, Alzoubi K, Chatterjee M, Walker B, Münzer P, Luo D, Umbach AT, Elvira B, Chen H, Voelkl J, Föller M, Mak TW, Borst O, Gawaz M, and Lang F
- Subjects
- Animals, Apoptosis, Blood Coagulation, Calcium Channels metabolism, Calcium Signaling, Caspase 3 metabolism, Cell Degranulation, Chlorides, Disease Models, Animal, Female, Ferric Compounds, Genotype, Hyaluronan Receptors blood, Hyaluronan Receptors genetics, Male, Mice, Knockout, ORAI1 Protein, Phenotype, Phospholipids blood, Platelet Glycoprotein GPIIb-IIIa Complex metabolism, Regional Blood Flow, Selenoprotein P metabolism, Stress, Mechanical, Thrombin metabolism, Thrombosis blood, Thrombosis genetics, Blood Platelets metabolism, Cell Membrane metabolism, Hyaluronan Receptors metabolism, Mechanotransduction, Cellular, Phospholipids metabolism, Platelet Adhesiveness, Thrombosis metabolism
- Abstract
CD44 is required for signalling of macrophage migration inhibitory factor (MIF), an anti-apoptotic pro-inflammatory cytokine. MIF is expressed and released from blood platelets, key players in the orchestration of occlusive vascular disease. Nothing is known about a role of CD44 in the regulation of platelet function. The present study thus explored whether CD44 modifies degranulation (P-selectin exposure), integrin activation, caspase activity, phosphatidylserine exposure on the platelet surface, platelet volume, Orai1 protein abundance and cytosolic Ca(2+)-activity ([Ca2+]i). Platelets from mice lacking CD44 (cd44(-/-)) were compared to platelets from corresponding wild-type mice (cd44(+/+)). In resting platelets, P-selectin abundance, α(IIb)β3 integrin activation, caspase-3 activity and phosphatidylserine exposure were negligible in both genotypes and Orai1 protein abundance, [Ca2+]i, and volume were similar in cd44(-/-) and cd44(+/+) platelets. Platelet degranulation and α(IIb)β3 integrin activation were significantly increased by thrombin (0.02 U/ml), collagen related peptide (CRP, 2 µg/ml and Ca(2+)-store depletion with thapsigargin (1 µM), effects more pronounced in cd44(-/-) than in cd44(+/+) platelets. Thrombin (0.02 U/ml) increased platelet [Ca2+]i, caspase-3 activity, phosphatidylserine exposure and Orai1 surface abundance, effects again significantly stronger in cd44(-/-) than in cd44(+/+) platelets. Thrombin further decreased forward scatter in cd44(-/-) and cd44(+/+) platelets, an effect which tended to be again more pronounced in cd44(-/-) than in cd44(+/+) platelets. Platelet adhesion and in vitro thrombus formation under high arterial shear rates (1,700 s(-1)) were significantly augmented in cd44(-/-) mice. In conclusion, genetic deficiency of CD44 augments activation, apoptosis and pro-thrombotic potential of platelets.
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- 2016
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11. Platelet-derived HMGB1 is a critical mediator of thrombosis.
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Vogel S, Bodenstein R, Chen Q, Feil S, Feil R, Rheinlaender J, Schäffer TE, Bohn E, Frick JS, Borst O, Münzer P, Walker B, Markel J, Csanyi G, Pagano PJ, Loughran P, Jessup ME, Watkins SC, Bullock GC, Sperry JL, Zuckerbraun BS, Billiar TR, Lotze MT, Gawaz M, and Neal MD
- Subjects
- Animals, Blood Platelets pathology, Cell Membrane genetics, Guanylate Cyclase genetics, Guanylate Cyclase metabolism, HMGB1 Protein genetics, Mice, Mice, Transgenic, Myeloid Differentiation Factor 88 genetics, Myeloid Differentiation Factor 88 metabolism, Thrombosis genetics, Thrombosis pathology, Toll-Like Receptor 4 genetics, Toll-Like Receptor 4 metabolism, Blood Coagulation, Blood Platelets metabolism, Cell Membrane metabolism, HMGB1 Protein metabolism, Platelet Aggregation, Thrombosis metabolism
- Abstract
Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.
- Published
- 2015
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12. Impact of the serum- and glucocorticoid-inducible kinase 1 on platelet dense granule biogenesis and secretion.
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Walker B, Schmid E, Russo A, Schmidt EM, Burk O, Münzer P, Velic A, Macek B, Schaller M, Schwab M, Seabra MC, Gawaz M, Lang F, and Borst O
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- Adenosine Triphosphate blood, Adenosine Triphosphate metabolism, Animals, Blood Platelets metabolism, Blood Platelets ultrastructure, Carotid Artery Injuries blood, Carotid Artery Injuries genetics, Carotid Artery Injuries pathology, Cells, Cultured, Cytoplasmic Granules metabolism, Cytoplasmic Granules ultrastructure, Disease Models, Animal, Female, Genotype, Immediate-Early Proteins deficiency, Immediate-Early Proteins genetics, Male, Megakaryocytes enzymology, Megakaryocytes metabolism, Mice, Knockout, Phenotype, Platelet Aggregation, Protein Serine-Threonine Kinases deficiency, Protein Serine-Threonine Kinases genetics, RNA, Messenger metabolism, Secretory Vesicles metabolism, Secretory Vesicles ultrastructure, Serotonin blood, Serotonin metabolism, Signal Transduction, Tetraspanin 30 blood, Tetraspanin 30 metabolism, Thrombosis blood, Thrombosis genetics, Thrombosis pathology, Time Factors, Transfection, Up-Regulation, rab GTP-Binding Proteins genetics, rab GTP-Binding Proteins metabolism, Blood Platelets enzymology, Carotid Artery Injuries enzymology, Cytoplasmic Granules enzymology, Immediate-Early Proteins blood, Platelet Activation, Protein Serine-Threonine Kinases blood, Secretory Vesicles enzymology, Thrombosis enzymology
- Abstract
Background: Platelet secretion is critical to development of acute thrombotic occlusion. Platelet dense granules contain a variety of important hemostatically active substances. Nevertheless, biogenesis of platelet granules is poorly understood., Objectives: Serum- and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be highly expressed in platelets and megakaryocytes, but its role in the regulation of platelet granule biogenesis and its impact on thrombosis has not been investigated so far., Methods and Results: Electron microscopy analysis of the platelet ultrastructure revealed a significant reduction in the number and packing of dense granules in platelets lacking SGK1 (sgk1(-/-) ). In sgk1(-/-) platelets serotonin content was significantly reduced and activation-dependent secretion of ATP, serotonin and CD63 significantly impaired. In vivo adhesion after carotis ligation was significantly decreased in platelets lacking SGK1 and occlusive thrombus formation after FeCl3 -induced vascular injury was significantly diminished in sgk1(-/-) mice. Transcript levels and protein abundance of dense granule biogenesis regulating GTPase Rab27b were significantly reduced in sgk1(-/-) platelets without affecting Rab27b mRNA stability. In MEG-01 cells transfection with constitutively active (S422) (D) SGK1 but not with inactive (K127) (N) SGK1 significantly enhanced Rab27b mRNA levels. Sgk1(-/-) megakaryocytes show significantly reduced expression of Rab27b and serotonin/CD63 levels compared with sgk1(+/+) megakaryocytes. Proteome analysis identified nine further vesicular transport proteins regulated by SGK1, which may have an impact on impaired platelet granule biogenesis in sgk1(-/-) platelets independent of Rab27b., Conclusions: The present observations identify SGK1 as a novel powerful regulator of platelet dense granule biogenesis, platelet secretion and thrombus formation. SGK1 is at least partially effective because it regulates transcription of Rab27b in megakaryocytes., (© 2015 International Society on Thrombosis and Haemostasis.)
- Published
- 2015
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13. Loss of oligophrenin1 leads to uncontrolled Rho activation and increased thrombus formation in mice.
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Fotinos A, Klier M, Gowert NS, Münzer P, Klatt C, Beck S, Borst O, Billuart P, Schaller M, Lang F, Gawaz M, and Elvers M
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- Animals, Cytoskeletal Proteins genetics, Cytoskeleton enzymology, Disease Models, Animal, Enzyme Activation, Female, GTPase-Activating Proteins genetics, Male, Mice, Inbred C57BL, Mice, Knockout, Neuropeptides blood, Nuclear Proteins genetics, Platelet Activation, Pseudopodia enzymology, Signal Transduction, Thrombosis blood, Thrombosis genetics, Time Factors, cdc42 GTP-Binding Protein blood, rac1 GTP-Binding Protein blood, rhoA GTP-Binding Protein, Blood Coagulation, Blood Platelets enzymology, Cytoskeletal Proteins deficiency, GTPase-Activating Proteins deficiency, Nuclear Proteins deficiency, Thrombosis enzymology, rho GTP-Binding Proteins blood
- Abstract
Background: Platelet cytoskeletal reorganization is essential for platelet adhesion and thrombus formation in hemostasis and thrombosis. The Rho GTPases RhoA, Rac1 and Cdc42 are the main players in cytoskeletal dynamics of platelets and induce filopodia and lamellipodia formation and actin polymerization to strongly increase the platelet surface upon activation. Moreover, they are important for platelet secretion, integrin activation and arterial thrombus formation., Objectives: Rho GTPases are regulated by GTPase-activating proteins (GAPs) that stimulate their GTPase activity to terminate Rho signaling. The regulation of Rho GTPase activity in platelets is not well defined. Recently, we identified oligophrenin1 (OPHN1), a RhoGAP in platelets that exhibits strong GTPase-stimulating activity towards RhoA, Cdc42 and Rac1., Results: In the present study we show for the first time, that deficiency of OPHN1 led to abnormal Rho activation and increased platelet cytoskeletal reorganization, including cell adhesion and lamellipodia formation on fibrinogen. Furthermore, platelets from ophn1(-/-) mice showed enhanced susceptibility to platelet activation with alterations in actin distribution and early release of granules. Platelet activation was enhanced following GPVI and PAR4 stimulation. This translated into elevated platelet thrombus formation and promoted arterial thrombosis under low shear conditions with altered hemostasis, as detected by tail bleeding time., Conclusions: The results of the present study identified OPHN1 as an important regulator of platelet cytoskeletal reorganization and demonstrate that abnormal regulation of Rho proteins leads to increased platelet adhesion and thrombus formation under low shear conditions in vitro and in vivo, suggesting a prothrombotic phenotype of mice critical for acute thrombotic occlusions., (© 2014 International Society on Thrombosis and Haemostasis.)
- Published
- 2015
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14. Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo.
- Author
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Seizer P, Ungern-Sternberg SN, Schönberger T, Borst O, Münzer P, Schmidt EM, Mack AF, Heinzmann D, Chatterjee M, Langer H, Malešević M, Lang F, Gawaz M, Fischer G, and May AE
- Subjects
- Animals, Blood Platelets drug effects, Carotid Artery Injuries blood, Carotid Artery Injuries enzymology, Carotid Artery Injuries genetics, Cell Degranulation drug effects, Chlorides, Cyclophilin A antagonists & inhibitors, Cyclophilin A genetics, Disease Models, Animal, Dose-Response Relationship, Drug, Ferric Compounds, Fibrinolytic Agents pharmacology, Humans, Mice, Inbred C57BL, Mice, Knockout, Platelet Aggregation drug effects, Platelet Aggregation Inhibitors pharmacology, Proto-Oncogene Proteins c-akt deficiency, Proto-Oncogene Proteins c-akt genetics, Thrombosis blood, Thrombosis chemically induced, Thrombosis genetics, Thrombosis prevention & control, Time Factors, Basigin blood, Blood Platelets enzymology, Cyclophilin A blood, Phosphatidylinositol 3-Kinases blood, Platelet Adhesiveness drug effects, Proto-Oncogene Proteins c-akt blood, Signal Transduction drug effects, Thrombosis enzymology
- Abstract
Objective: Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types. Whereas the important role of intracellular CyPA for platelet function has been reported, the effect of extracellular CyPA on platelet function has not been investigated yet., Approach and Results: Inhibition of extracellular CyPA through a novel specific inhibitor MM284 reduced thrombus after ferric chloride-induced injury in vivo. In vitro extracellular CyPA enhanced thrombus formation even in CyPA(-/-) platelets. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time- and dose-dependent manner. Inhibition of the platelet surface receptor extracellular matrix metalloproteinase inducer (cluster of differentiation 147) by an anticluster of differentiation 147 monoclonal antibody significantly reduced CyPA-dependent platelet degranulation. Pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by an anticluster of differentiation 147 monoclonal antibody (intravital microscopy). The role of extracellular CyPA in adhesion could be confirmed by infusing CyPA(-/-) platelets in CyPA(+/+) mice and by infusing CyPA(+/+) platelets in CyPA(-/-) mice. Stimulation of platelets with CyPA induced phosphorylation of Akt, which could in turn be inhibited in the presence of phosphoinositid-3-kinase inhibitors. Akt-1(-/-) platelets revealed a markedly decreased degranulation on CyPA stimulation. Finally, ADP-induced platelet aggregation was attenuated by MM284, as well as by inhibiting paracrine-secreted CyPA without directly affecting Ca(2+)-signaling., Conclusions: Extracellular CyPA activates platelets via cluster of differentiation 147-mediated phosphoinositid-3-kinase/Akt-signaling, leading to enhanced adhesion and thrombus formation independently of intracellular CyPA. Targeting extracellular CyPA via a specific inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA., (© 2014 American Heart Association, Inc.)
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- 2015
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15. Pre-activated blood platelets and a pro-thrombotic phenotype in APP23 mice modeling Alzheimer's disease.
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Jarre A, Gowert NS, Donner L, Münzer P, Klier M, Borst O, Schaller M, Lang F, Korth C, and Elvers M
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- Alzheimer Disease, Animals, Blood Platelets metabolism, Cell Adhesion drug effects, Disease Models, Animal, Glycoproteins metabolism, Integrins metabolism, Megakaryocytes cytology, Mice, Mice, Transgenic, P-Selectin metabolism, Phenotype, Platelet Activation, Thrombin pharmacology, Thrombosis metabolism, Thrombosis pathology, Blood Platelets cytology
- Abstract
Platelet activation and thrombus formation play a critical role in primary hemostasis but also represent a pathophysiological mechanism leading to acute thrombotic vascular occlusions. Besides, platelets modulate cellular processes including inflammation, angiogenesis and neurodegeneration. On the other hand, platelet activation and thrombus formation are altered in different diseases leading to either bleeding complications or pathological thrombus formation. For many years platelets have been considered to play a role in neuroinflammatory diseases such as Alzheimer's disease (AD). AD is characterized by deposits of amyloid-β (Aβ) and strongly related to vascular diseases with platelets playing a critical role in the progression of AD because exposure of platelets to Aβ induces platelet activation, platelet Aβ release, and enhanced platelet adhesion to collagen in vitro and at the injured carotid artery in vivo. However, the molecular mechanisms and the relation between vascular pathology and amyloid-β plaque formation in the pathogenesis of AD are not fully understood. Compelling evidence is suggestive for altered platelet activity in AD patients. Thus we analyzed platelet activation and thrombus formation in aged AD transgenic mice (APP23) known to develop amyloid-β deposits in the brain parenchyma and cerebral vessels. As a result, platelets are in a pre-activated state in blood of APP23 mice and showed strongly enhanced integrin activation, degranulation and spreading kinetics on fibrinogen surfaces upon stimulation. This enhanced platelet signaling translated into almost unlimited thrombus formation on collagen under flow conditions in vitro and accelerated vessel occlusion in vivo suggesting that these mice are at high risk of arterial thrombosis leading to cerebrovascular and unexpectedly to cardiovascular complications that might be also relevant in AD patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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16. 1,25(OH)2 vitamin D3-dependent inhibition of platelet Ca2+ signaling and thrombus formation in klotho-deficient mice.
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Borst O, Münzer P, Schmid E, Schmidt EM, Russo A, Walker B, Yang W, Leibrock C, Szteyn K, Schmidt S, Elvers M, Faggio C, Shumilina E, Kuro-o M, Gawaz M, and Lang F
- Subjects
- Animals, Calcium Channels metabolism, Cell Nucleus metabolism, Cytosol metabolism, Down-Regulation, Klotho Proteins, Megakaryocytes cytology, Membrane Glycoproteins metabolism, Mice, Mice, Transgenic, NF-kappa B metabolism, ORAI1 Protein, Patch-Clamp Techniques, Platelet Aggregation, Signal Transduction, Stromal Interaction Molecule 1, Transfection, Blood Platelets metabolism, Calcitriol metabolism, Calcium metabolism, Calcium Signaling, Glucuronidase genetics, Thrombosis metabolism
- Abstract
Platelets are activated by increased cytosolic Ca(2+) concentration ([Ca(2+)]i) following store-operated calcium entry (SOCE) accomplished by calcium-release-activated calcium (CRAC) channel moiety Orai1 and its regulator STIM1. In other cells, Ca(2+) transport is regulated by 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 formation is inhibited by klotho and excessive in klotho-deficient mice (kl/kl). The present study explored the effect of klotho deficiency on platelet Ca(2+) signaling and activation. Platelets and megakaryocytes isolated from WT and kl/kl-mice were analyzed by RT-PCR, Western blotting, confocal microscopy, Fura-2-fluorescence, patch clamp, flow cytometry, aggregometry, and flow chamber. STIM1/Orai1 transcript and protein levels, SOCE, agonist-induced [Ca(2+)]i increase, activation-dependent degranulation, integrin αIIbβ3 activation and aggregation, and thrombus formation were significantly blunted in kl/kl platelets (by 27-90%). STIM1/Orai1 transcript and protein levels, as well as CRAC currents, were significantly reduced in kl/kl megakaryocytes (by 38-73%) and 1,25(OH)2D3-treated WT megakaryocytes. Nuclear NF-κB subunit p50/p65 abundance was significantly reduced in kl/kl-megakaryocytes (by 51-76%). Transfection with p50/p65 significantly increased STIM1/Orai1 transcript and protein levels in megakaryocytic MEG-01 cells (by 46-97%). Low-vitamin D diet (LVD) of kl/kl mice normalized plasma 1,25(OH)2D3 concentration and function of platelets and megakaryocytes. Klotho deficiency inhibits platelet Ca(2+) signaling and activation, an effect at least partially due to 1,25(OH)2D3-dependent down-regulation of NF-κB activity and STIM1/Orai1 expression in megakaryocytes.
- Published
- 2014
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17. Blood platelets in the progression of Alzheimer's disease.
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Gowert NS, Donner L, Chatterjee M, Eisele YS, Towhid ST, Münzer P, Walker B, Ogorek I, Borst O, Grandoch M, Schaller M, Fischer JW, Gawaz M, Weggen S, Lang F, Jucker M, and Elvers M
- Subjects
- Adult, Alzheimer Disease blood, Alzheimer Disease genetics, Amyloid ultrastructure, Amyloid beta-Peptides pharmacology, Animals, Apoptosis drug effects, Blood Platelets drug effects, Blood Platelets ultrastructure, Cells, Cultured, Cerebral Amyloid Angiopathy blood, Cerebral Amyloid Angiopathy genetics, Cerebral Amyloid Angiopathy metabolism, Disease Models, Animal, Disease Progression, Humans, Mice, Inbred C57BL, Mice, Transgenic, Microscopy, Electron, Microscopy, Fluorescence, Middle Aged, Platelet Activation drug effects, Reactive Oxygen Species metabolism, Young Adult, Alzheimer Disease metabolism, Amyloid metabolism, Amyloid beta-Peptides metabolism, Blood Platelets metabolism
- Abstract
Alzheimer's disease (AD) is characterized by neurotoxic amyloid-ß plaque formation in brain parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA). Besides CAA, AD is strongly related to vascular diseases such as stroke and atherosclerosis. Cerebrovascular dysfunction occurs in AD patients leading to alterations in blood flow that might play an important role in AD pathology with neuronal loss and memory deficits. Platelets are the major players in hemostasis and thrombosis, but are also involved in neuroinflammatory diseases like AD. For many years, platelets were accepted as peripheral model to study the pathophysiology of AD because platelets display the enzymatic activities to generate amyloid-ß (Aß) peptides. In addition, platelets are considered to be a biomarker for early diagnosis of AD. Effects of Aß peptides on platelets and the impact of platelets in the progression of AD remained, however, ill-defined. The present study explored the cellular mechanisms triggered by Aß in platelets. Treatment of platelets with Aß led to platelet activation and enhanced generation of reactive oxygen species (ROS) and membrane scrambling, suggesting enhanced platelet apoptosis. More important, platelets modulate soluble Aß into fibrillar structures that were absorbed by apoptotic but not vital platelets. This together with enhanced platelet adhesion under flow ex vivo and in vivo and platelet accumulation at amyloid deposits of cerebral vessels of AD transgenic mice suggested that platelets are major contributors of CAA inducing platelet thrombus formation at vascular amyloid plaques leading to vessel occlusion critical for cerebrovascular events like stroke.
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- 2014
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18. Dynamic adhesion of eryptotic erythrocytes to immobilized platelets via platelet phosphatidylserine receptors.
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Walker B, Towhid ST, Schmid E, Hoffmann SM, Abed M, Münzer P, Vogel S, Neis F, Brucker S, Gawaz M, Borst O, and Lang F
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- Adenosine Diphosphate metabolism, Annexin A5 metabolism, CD36 Antigens immunology, Cell Adhesion, Cell Membrane metabolism, Chemokine CXCL16, Chemokines, CXC immunology, Glucose deficiency, Glucose metabolism, Humans, Platelet Adhesiveness, Receptors, Cell Surface metabolism, Receptors, Scavenger immunology, Thrombin metabolism, Apoptosis, Blood Platelets metabolism, CD36 Antigens metabolism, Chemokines, CXC metabolism, Erythrocytes metabolism, Phosphatidylserines metabolism, Receptors, Scavenger metabolism
- Abstract
Glucose depletion of erythrocytes triggers suicidal erythrocyte death or eryptosis, which leads to cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to endothelial cells by a mechanism involving phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 at the endothelial cell membrane. Nothing has hitherto been known about an interaction between eryptotic erythrocytes and platelets, the decisive cells in primary hemostasis and major players in thrombotic vascular occlusion. The present study thus explored whether and how glucose-depleted erythrocytes adhere to platelets. To this end, adhesion of phosphatidylserine-exposing erythrocytes to platelets under flow conditions was examined in a flow chamber model at arterial shear rates. Platelets were immobilized on collagen and further stimulated with adenosine diphosphate (ADP, 10 μM) or thrombin (0.1 U/ml). As a result, a 48-h glucose depletion triggered phosphatidylserine translocation to the erythrocyte surface and augmented the adhesion of erythrocytes to immobilized platelets, an effect significantly increased upon platelet stimulation. Adherence of erythrocytes to platelets was blunted by coating of erythrocytic phosphatidylserine with annexin V or by neutralization of platelet phosphatidylserine receptors CXCL16 and CD36 with respective antibodies. In conclusion, glucose-depleted erythrocytes adhere to platelets. The adhesive properties of platelets are augmented by platelet activation. Erythrocyte adhesion to immobilized platelets requires phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 expression on platelets. Thus platelet-mediated erythrocyte adhesion may foster thromboocclusive complications in diseases with stimulated phosphatidylserine exposure of erythrocytes.
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- 2014
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19. Acid sphingomyelinase regulates platelet cell membrane scrambling, secretion, and thrombus formation.
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Münzer P, Borst O, Walker B, Schmid E, Feijge MA, Cosemans JM, Chatterjee M, Schmidt EM, Schmidt S, Towhid ST, Leibrock C, Elvers M, Schaller M, Seizer P, Ferlinz K, May AE, Gulbins E, Heemskerk JW, Gawaz M, and Lang F
- Subjects
- Adenosine Triphosphate blood, Animals, Blood Platelets drug effects, Calcium blood, Cell Membrane drug effects, Ceramides blood, Chlorides, Disease Models, Animal, Enzyme Inhibitors pharmacology, Female, Ferric Compounds, Fibrinolytic Agents pharmacology, Male, Mice, Mice, Knockout, P-Selectin blood, Phosphatidylserines blood, Platelet Aggregation, Platelet Glycoprotein GPIIb-IIIa Complex metabolism, Sphingomyelin Phosphodiesterase antagonists & inhibitors, Sphingomyelin Phosphodiesterase deficiency, Sphingomyelin Phosphodiesterase genetics, Thrombin metabolism, Thrombosis blood, Thrombosis chemically induced, Thrombosis genetics, Thrombosis prevention & control, Time Factors, Blood Platelets enzymology, Cell Degranulation drug effects, Cell Membrane enzymology, Platelet Activation drug effects, Sphingomyelin Phosphodiesterase blood, Thrombosis enzymology
- Abstract
Objective: Platelet activation is essential for primary hemostasis and acute thrombotic vascular occlusions. On activation, platelets release their prothrombotic granules and expose phosphatidylserine, thus fostering thrombin generation and thrombus formation. In other cell types, both degranulation and phosphatidylserine exposure are modified by sphingomyelinase-dependent formation of ceramide. The present study thus explored whether acid sphingomyelinase participates in the regulation of platelet secretion, phosphatidylserine exposure, and thrombus formation., Approach and Results: Collagen-related peptide-induced or thrombin-induced ATP release and P-selectin exposure were significantly blunted in platelets from Asm-deficient mice (Smpd1(-/-)) when compared with platelets from wild-type mice (Smpd1(+/+)). Moreover, phosphatidylserine exposure and thrombin generation were significantly less pronounced in Smpd1(-/-) platelets than in Smpd1(+/+) platelets. In contrast, platelet integrin αIIbβ3 activation and aggregation, as well as activation-dependent Ca(2+) flux, were not significantly different between Smpd1(-/-) and Smpd1(+/+) platelets. In vitro thrombus formation at shear rates of 1700 s(-1) and in vivo thrombus formation after FeCl3 injury were significantly blunted in Smpd1(-/-) mice while bleeding time was unaffected. Asm-deficient platelets showed significantly reduced activation-dependent ceramide formation, whereas exogenous ceramide rescued diminished platelet secretion and thrombus formation caused by Asm deficiency. Treatment of Smpd1(+/+) platelets with bacterial sphingomyelinase (0.01 U/mL) increased, whereas treatment with functional acid sphingomyelinase-inhibitors, amitriptyline or fluoxetine (5 μmol/L), blunted activation-dependent platelet degranulation, phosphatidylserine exposure, and thrombus formation. Impaired degranulation and thrombus formation of Smpd1(-/-) platelets were again overcome by exogenous bacterial sphingomyelinase., Conclusions: Acid sphingomyelinase is a completely novel element in the regulation of platelet plasma membrane properties, secretion, and thrombus formation.
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- 2014
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20. Regulation of STIM1/Orai1-dependent Ca2+ signalling in platelets.
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Lang F, Münzer P, Gawaz M, and Borst O
- Subjects
- Animals, Calcium Signaling, Humans, Immediate-Early Proteins metabolism, NF-kappa B metabolism, ORAI1 Protein, Platelet Activation, Protein Serine-Threonine Kinases metabolism, Reactive Oxygen Species metabolism, Stromal Interaction Molecule 1, Blood Platelets metabolism, Calcium Channels metabolism, Diabetes Mellitus, Type 2 immunology, Membrane Proteins metabolism, Metabolic Syndrome immunology, Neoplasm Proteins metabolism
- Abstract
Platelet secretion and aggregation as well as thrombus formation of blood platelets critically depend on increase of cytosolic Ca2+ concentration ([Ca2+]i) mainly resulting from intracellular Ca2+ release followed by store operated Ca2+ entry (SOCE) through Ca2+ release activated channels (CRAC). SOCE is in part accomplished by the pore forming unit Orai and its regulator stromal interaction molecule (STIM). Orai1 and STIM1 transcription is stimulated by NF-κB (nuclear factor kappa B). Serum- and glucocorticoid-inducible kinase 1 (SGK1) up-regulates NF-κB-activity in megakaryocytes and thus Orai1-expression and SOCE in platelets. SGK1 is thus a powerful regulator of platelet Ca2+-signalling and thrombus formation and presumably participates in the regulation of platelet activation by a variety of hormones as well as clinical conditions (e.g. type 2 diabetes or metabolic syndrome) associated with platelet hyperaggregability and increased risk of thromboocclusive events. SOCE in platelets is further regulated by scaffolding protein Homer and chaperone protein cyclophilin A (CyPA). Additional potential regulators of Orai1/STIM1 and thus SOCE in platelets include AMP activated kinase (AMPK), protein kinase A (PKA), reactive oxygen species, lipid rafts, pH and mitochondrial Ca2+ buffering. Future studies are required defining the significance of those mechanisms for platelet Orai1 abundance and function, for SOCE into platelets and for platelet function in cardiovascular diseases.
- Published
- 2013
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21. Increased platelet Ca2+ channel Orai1 expression upon platelet activation and in patients with acute myocardial infarction.
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Tolios A, Gatidis S, Münzer P, Liu G, Towhid ST, Karathanos A, Tavlaki E, Geisler T, Seizer P, May AE, Bigalke B, Borst O, Gawaz M, and Lang F
- Subjects
- Aged, Angina, Stable blood, C-Reactive Protein metabolism, C-Reactive Protein pharmacology, Cell Membrane metabolism, Female, Humans, Male, Middle Aged, Myocardial Infarction etiology, ORAI1 Protein, Platelet Activation drug effects, Platelet Activation physiology, Risk Factors, Blood Platelets metabolism, Calcium Channels blood, Myocardial Infarction blood
- Published
- 2013
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22. Chorein sensitivity of cytoskeletal organization and degranulation of platelets.
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Schmidt EM, Schmid E, Münzer P, Hermann A, Eyrich AK, Russo A, Walker B, Gu S, vom Hagen JM, Faggio C, Schaller M, Föller M, Schöls L, Gawaz M, Borst O, Storch A, Stournaras C, and Lang F
- Subjects
- Actins metabolism, Adult, Blood Platelets physiology, Blood Platelets ultrastructure, Blotting, Western, Cell Line, Tumor, Class Ia Phosphatidylinositol 3-Kinase metabolism, Female, Humans, Male, Microscopy, Confocal, Microscopy, Electron, Transmission, Middle Aged, Neuroacanthocytosis blood, Neuroacanthocytosis genetics, Neuroacanthocytosis metabolism, Phosphorylation, Platelet Aggregation, R-SNARE Proteins metabolism, RNA Interference, Vesicular Transport Proteins genetics, Young Adult, p21-Activated Kinases metabolism, Blood Platelets metabolism, Cell Degranulation, Cytoskeleton metabolism, Vesicular Transport Proteins metabolism
- Abstract
Chorea-acanthocytosis (ChAc), a lethal disease caused by defective chorein, is characterized by neurodegeneration and erythrocyte acanthocytosis. The functional significance of chorein in other cell types remained ill-defined. The present study revealed chorein expression in blood platelets. As compared to platelets from healthy volunteers, platelets from patients with ChAc displayed a 47% increased globular/filamentous actin ratio, indicating actin depolymerization. Moreover, phosphoinositide-3-kinase subunit p85 phosphorylation, p21 protein-activated kinase (PAK1) phosphorylation, as well as vesicle-associated membrane protein 8 (VAMP8) expression were significantly reduced in platelets from patients with ChAc (by 17, 22, and 39%, respectively) and in megakaryocytic (MEG-01) cells following chorein silencing (by 16, 54, and 11%, respectively). Activation-induced platelet secretion from dense granules (ATP release) and α granules (P-selectin exposure) were significantly less (by 55% after stimulation with 1 μg/ml CRP and by 33% after stimulation with 5 μM TRAP, respectively) in ChAc platelets than in control platelets. Furthermore, platelet aggregation following stimulation with different platelet agonists was significantly impaired. These observations reveal a completely novel function of chorein, i.e., regulation of secretion and aggregation of blood platelets.
- Published
- 2013
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23. Stimulation of platelet apoptosis by balhimycin.
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Towhid ST, Tolios A, Münzer P, Schmidt EM, Borst O, Gawaz M, Stegmann E, and Lang F
- Subjects
- Adult, Anti-Bacterial Agents administration & dosage, Apoptosis drug effects, Apoptosis physiology, Blood Platelets drug effects, Cell Size, Cells, Cultured, Dose-Response Relationship, Drug, Female, Humans, Male, Middle Aged, Vancomycin administration & dosage, Blood Platelets physiology, Calcium metabolism, Ceramides metabolism, Membrane Fluidity drug effects, Membrane Fluidity physiology, Vancomycin analogs & derivatives
- Abstract
Glycopeptides, such as vancomycin, are powerful antibiotics against methicillin-resistant Staphylococcus aureus. Balhimycin, a glycopeptide antibiotic isolated from Amycolatopsis balhimycina, is similarly effective as vancomycin. Side effects of vancomycin include triggering of platelet apoptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine exposure at the cell surface. Stimulation of apoptosis may involve increase of cytosolic Ca(2+) activity, ceramide formation, mitochondrial depolarization and/or caspase activation. An effect of balhimycin on apoptosis has, however, never been reported. The present study thus tested whether balhimycin triggers platelet apoptosis. Human blood platelets were treated with balhimycin and cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V-binding, cytosolic Ca(2+) activity from fluo-3AM fluorescence, ceramide formation utilizing antibodies, mitochondrial potential from DiOC6 fluorescence, and caspase-3 activity utilizing antibodies. As a result, a 30 min exposure to balhimycin significantly decreased cell volume (≥1 μg/ml), triggered annexin V binding (≥1 μg/ml), increased cytosolic Ca(2+) activity (≥1 μg/ml), stimulated ceramide formation (≥10 μg/ml), depolarized mitochondria (≥1 μg/ml) and activated caspase-3 (≥1 μg/ml). Cell membrane scrambling and caspase-3 activation were virtually abrogated by removal of extracellular Ca(2+). Cell membrane scrambling was not significantly blunted by pancaspase inhibition with zVAD-FMK (1μM). In conclusion, balhimycin triggers cell membrane scrambling of platelets, an effect dependent on Ca(2+), but not on activation of caspases., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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24. Skepinone-L, a novel potent and highly selective inhibitor of p38 MAP kinase, effectively impairs platelet activation and thrombus formation.
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Borst O, Walker B, Münzer P, Russo A, Schmid E, Faggio C, Bigalke B, Laufer S, Gawaz M, and Lang F
- Subjects
- 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid pharmacology, Adenosine Triphosphate metabolism, Blood Platelets metabolism, Calcium metabolism, Carrier Proteins pharmacology, Fura-2 chemistry, HSP27 Heat-Shock Proteins metabolism, Humans, Peptides pharmacology, Phospholipases A2 metabolism, Phosphorylation drug effects, Platelet Activation drug effects, Platelet Aggregation drug effects, Shear Strength drug effects, Thrombin pharmacology, Thrombosis metabolism, Thrombosis pathology, Thromboxane B2 metabolism, p38 Mitogen-Activated Protein Kinases metabolism, Blood Platelets drug effects, Dibenzocycloheptenes pharmacology, Protein Kinase Inhibitors pharmacology, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors
- Abstract
Background/aims: Platelets are critically important for primary haemostasis and the major players in thrombotic vascular occlusion. Platelets are activated by agonists, such as thrombin and collagen-related peptide as well as second-wave mediators including thromboxane A2 via different intracellular signaling pathways resulting in degranulation, aggregation and thrombus formation. Platelet activation is paralleled by phosphorylation and activation of p38 MAPK. The limited specificity of hitherto known p38 MAPK inhibitors precluded safe conclusions on the precise role of p38 MAPK in the regulation of platelet function. The present study examined the impact of Skepinone-L, a novel and highly selective inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), on platelet activation and thrombus formation., Methods: Experiments were performed in freshly isolated human platelets. Protein phosphorylation was quantified by Western blotting, thromboxane B2 synthesis by enzyme immunoassay, ATP release by ChronoLume luciferin assay, cytosolic Ca(2+) concentration by Fura-2 fluorescence-measurements, platelet aggregation by a light transmissions measurement and in vitro thrombus formation by a flow chamber., Results: Skepinone-L (1 μM) virtually abrogated the phosphorylation of platelet p38 MAPK substrate Hsp27 following stimulation with CRP (1 μg/ml), thrombin (5 mU/ml) or thromboxane A2 analogue U-46619 (1 μM). Furthermore, Skepinone-L significantly blunted activation-dependent platelet secretion and aggregation following threshold concentrations of CRP, thrombin and thromboxane A2 analogue U-46619. Skepinone-L did not impair platelet Ca(2+) signaling but prevented agonist-induced thromboxane A2 synthesis through abrogation of p38 MAPK-dependent phosphorylation of platelet cytosolic phospholipase A2 (cPLA2). Skepinone-L further markedly blunted thrombus formation under low (500-s) and high (1700-s) arterial shear rates., Conclusions: The present study discloses a powerful inhibiting effect of p38 MAPK-blocker Skepinone-L on platelet activation and thrombus formation., (Copyright © 2013 S. Karger AG, Basel.)
- Published
- 2013
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25. Stimulation of platelet death by vancomycin.
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Towhid ST, Schmidt EM, Tolios A, Münzer P, Schmid E, Borst O, Gawaz M, Stegmann E, and Lang F
- Subjects
- Adult, Amino Acid Chloromethyl Ketones pharmacology, Aniline Compounds chemistry, Annexin A5 metabolism, Blood Platelets cytology, Calcium metabolism, Caspase 3 metabolism, Caspase Inhibitors pharmacology, Cell Size drug effects, Ceramides metabolism, Humans, Membrane Potential, Mitochondrial drug effects, Middle Aged, P-Selectin metabolism, Phosphatidylserines pharmacology, Platelet Glycoprotein GPIIb-IIIa Complex metabolism, Protein Binding, Thromboxane B2 metabolism, Xanthenes chemistry, Anti-Bacterial Agents pharmacology, Apoptosis drug effects, Blood Platelets drug effects, Vancomycin pharmacology
- Abstract
Background/aims: Side effects of vancomycin, a widely used antibiotic, include thrombocytopenia. The vancomycin-induced thrombocytopenia has been attributed to immune reactions. At least in theory, thrombocytopenia could result in part from the triggering of apoptosis, which results in cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. The cell membrane scrambling could be initiated by a signaling involving increase of cytosolic Ca(2+) activity, ceramide formation, mitochondrial depolarization and/or caspase activation. Vancomycin has indeed been shown to trigger neutrophil apoptosis. An effect of vancomycin on platelet apoptosis has, however, never been tested. The present study thus explored the effect of vancomycin on platelet activation and apoptosis., Methods: Human blood platelets were exposed to vancomycin and forward scatter was utilized to estimate cell volume, annexin V-binding to quantify phosphatidylserine (PS) exposure, Fluo-3 AM fluorescence to estimate cytosolic Ca(2+) activity ([Ca(2+)]i), antibodies to quantify ceramide formation and immunofluorescence to quantify protein abundance of active caspase-3., Results: A 30 minutes exposure to vancomycin (≥1 µg/ ml) decreased cell volume, triggered annexin V-binding, increased [Ca(2+)]i, activated caspase 3, stimulated ceramide formation, triggered release of thromboxane B2, and upregulated surface expression of CD62P (P-selectin) as well as activated integrin αllbβ3. Annexin V-binding and upregulation of CD62P (P-selectin) and integrin αllbβ3 was significantly blunted by removal of extracellular Ca(2+). Annexin V-binding was not significantly blunted by pan-caspase inhibitor zVAD-FMK (1 µM). In conclusion, vancomycin results in platelet activation and suicidal platelet death with increase of [Ca(2+)]i, caspase-3 activation, cell membrane scrambling and cell shrinkage. Activation and cell membrane scrambling required the presence of Ca(2+), but not activation of caspases., Conclusion: Vancomycin exposure leads to platelet activation and apoptosis., (Copyright © 2013 S. Karger AG, Basel.)
- Published
- 2013
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26. Stimulation of platelet apoptosis by peptidoglycan from Staphylococcus aureus 113.
- Author
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Towhid ST, Nega M, Schmidt EM, Schmid E, Albrecht T, Münzer P, Borst O, Götz F, and Lang F
- Subjects
- Amino Acid Chloromethyl Ketones pharmacology, Annexin A5 metabolism, Antibodies immunology, Caspase 3 metabolism, Caspase Inhibitors pharmacology, Cell Membrane drug effects, Cell Membrane metabolism, Cells, Cultured, Enzyme Activation, Humans, Integrin alpha2 biosynthesis, Mitochondria metabolism, Toll-Like Receptor 2 immunology, Apoptosis drug effects, Blood Platelets drug effects, Blood Platelets metabolism, Peptidoglycan pharmacology, Staphylococcus aureus chemistry
- Abstract
Peptidoglycan (PGN), a component of bacterial cell wall and belonging to "Microbe-Associated Molecular Patterns" (MAMP) triggers host reactions contributing to the pathophysiology of infectious disease. Host cell responses to PGN exposure include apoptosis. Bacterial infections may result in activation of blood platelets and thrombocytopenia. The present study explored, whether HPLC-purified fractions of PGNs from Staphylococcus aureus 113 triggers apoptosis of platelets. To this end platelets were exposed to PGN fractions and annexin-V binding determined to depict cell membrane scrambling, DiOC6 fluorescence to estimate depolarization of mitochondrial potential, Fluo-3AM staining for intracellular Ca(2+) activity ([Ca(2+)](i)) and immunofluorescence to quantify protein abundance of active caspase-3. As a result, a 30 min exposure to monomeric fraction (mPGN) (≥50 ng/ml) was followed by annexin-V binding, paralleled by increase of [Ca(2+)](i), mitochondrial depolarization, caspase-3 activation and integrin α(IIb)β(3) upregulation. The annexin-V binding was significantly blunted by anti-TLR-2 antibodies, in absence of extracellular Ca(2+), and by pancaspase inhibitor zVAD-FMK (1 μM). In conclusion, PGN triggers apoptosis of platelets in activation-dependent manner, characterized by mitochondrial depolarization, caspase-3 activation and cell membrane scrambling.
- Published
- 2012
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27. A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity.
- Author
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Elvers M, Grenegård M, Khoshjabinzadeh H, Münzer P, Borst O, Tian H, Di Paolo G, Lang F, Gawaz M, Lindahl TL, and Fälker K
- Subjects
- Animals, Biocatalysis, Domperidone analogs & derivatives, Domperidone pharmacology, Humans, Hydrolysis, Indoles pharmacology, Mice, Mice, Knockout, Phosphatidic Acids biosynthesis, Phosphatidic Acids chemistry, Phosphatidylcholines chemistry, Phosphatidylcholines metabolism, Phospholipase D antagonists & inhibitors, Phospholipase D deficiency, Blood Platelets metabolism, Phospholipase D metabolism
- Abstract
Platelet aggregation, secretion and thrombus formation play a critical role in primary hemostasis to prevent excessive blood loss. On the other hand, uncontrolled platelet activation leads to pathological thrombus formation resulting in myocardial infarction or stroke. Stimulation of heterotrimeric G-proteins by soluble agonists or immunoreceptor tyrosine based activation motif-coupled receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI lead to the activation of phospholipases that cleave membrane phospholipids to generate soluble second messengers. Platelets contain the phospholipases (PL) D1 and D2 which catalyze the hydrolysis of phosphatidylcholine to generate the second messenger phosphatidic acid (PA). The production of PA is abrogated by primary alcohols that have been widely used for the analysis of PLD-mediated processes. However, it is not clear if primary alcohols effectively reduce PA generation or if they induce PLD-independent cellular effects. In the present study we made use of the specific PLD inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) and show for the first time, that FIPI enhances platelet dense granule secretion and aggregation of human platelets. Further, FIPI has no effect on cytosolic Ca(2+) activity but needs proper Rho kinase signaling to mediate FIPI-induced effects on platelet activation. Upon FIPI treatment the phosphorylation of the PKC substrate pleckstrin was prominently enhanced suggesting that FIPI affects PKC-mediated secretion and aggregation in platelets. Similar effects of FIPI were observed in platelets from mouse wild-type and Pld1(-/-) mice pointing to a new role for PLD2 as a negative regulator of platelet sensitivity., (Copyright © 2012 Elsevier Inc. All rights reserved.)
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- 2012
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28. Translational regulation of the serum- and glucocorticoid-inducible kinase-1 (SGK1) in platelets.
- Author
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Pelzl L, Tolios A, Schmidt EM, Alesutan I, Walker B, Münzer P, Borst O, Gawaz M, and Lang F
- Subjects
- Androstadienes pharmacology, Calcium Channels biosynthesis, Cells, Cultured, Chromones pharmacology, Cytochalasin B pharmacology, Humans, Immediate-Early Proteins genetics, Morpholines pharmacology, ORAI1 Protein, Phosphodiesterase Inhibitors pharmacology, Phosphoinositide-3 Kinase Inhibitors, Platelet Activation drug effects, Platelet Activation genetics, Protein Biosynthesis drug effects, Protein Biosynthesis genetics, Protein Serine-Threonine Kinases genetics, Thrombin pharmacology, Transcription, Genetic, Wortmannin, Blood Platelets enzymology, Immediate-Early Proteins biosynthesis, Platelet Activation physiology, Protein Biosynthesis physiology, Protein Serine-Threonine Kinases biosynthesis, Thrombin physiology
- Abstract
Activation of platelets by thrombin opens pore forming channel protein Orai1 with subsequent store operated Ca(2+) entry (SOCE) and Ca(2+) dependent platelet granule release, integrin α(IIb)β(3) activation, adhesion, aggregation and thrombus formation. Orai1 and thus SOCE as well as platelet activation are up-regulated by the serum- and glucocorticoid-inducible kinase-1 (SGK1), which transcriptionally regulates Orai1 expression in megakaryocytes and thus determines Orai1 protein abundance in mature, circulating platelets. As platelets are devoid of nuclei, they are unable to modify protein abundance by regulation of transcription. However, they contain mRNA and thus could express novel protein by stimulation of protein translation. Translation is sensitive to actin polymerization and phosphoinositide-3-kinase (PI3K). Translational regulation of SGK1 expression has never been described before. The present study thus explored whether thrombin regulates SGK1 expression in platelets. As a result, according to RT-PCR mRNA encoding SGK1 is present in circulating platelets and significantly decreased by activation of platelets with thrombin (1 U/ml). The protein abundance of SGK1 is significantly enhanced by thrombin treatment, an effect significantly decreased by inhibition of translation with puromycin (100 nM) but not by inhibition of transcription with actinomycin (4 μg/ml). The increase of SGK1 protein abundance is blunted by inhibition of PI3K with wortmannin (100 nM) or LY294002 (25 μM), and by disruption of the cytoskeleton with cytochalasin B (1 μM). In conclusion, activation of platelets with thrombin stimulates the translation of SGK1., (Copyright © 2012 Elsevier Inc. All rights reserved.)
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- 2012
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29. Intracellular cyclophilin A is an important Ca(2+) regulator in platelets and critically involved in arterial thrombus formation.
- Author
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Elvers M, Herrmann A, Seizer P, Münzer P, Beck S, Schönberger T, Borst O, Martin-Romero FJ, Lang F, May AE, and Gawaz M
- Subjects
- Animals, CHO Cells, Calcium Signaling genetics, Cell Degranulation genetics, Cell Degranulation physiology, Cricetinae, Cricetulus, Cyclophilin A genetics, Cyclophilin A metabolism, Integrin beta3 metabolism, Intracellular Space metabolism, Mice, Mice, Knockout, Models, Biological, Peripheral Arterial Disease genetics, Peripheral Arterial Disease metabolism, Platelet Activation genetics, Thrombosis metabolism, Blood Platelets metabolism, Calcium metabolism, Cyclophilin A physiology, Thrombosis genetics
- Abstract
Platelet adhesion and aggregation play a critical role in primary hemostasis. Uncontrolled platelet activation leads to pathologic thrombus formation and organ failure. The decisive central step for different processes of platelet activation is the increase in cytosolic Ca(2+) activity ([Ca(2+)](i)). Activation-dependent depletion of intracellular Ca(2+) stores triggers Ca(2+) entry from the extracellular space. Stromal interaction molecule 1 (STIM1) has been identified as a Ca(2+) sensor that regulates store-operated Ca(2+) entry through activation of the pore-forming subunit Orai1, the major store-operated Ca(2+) entry channel in platelets. In the present study, we show for the first time that the chaperone protein cyclophilin A (CyPA) acts as a Ca(2+) modulator in platelets. CyPA deficiency strongly blunted activation-induced Ca(2+) mobilization from intracellular stores and Ca(2+) influx from the extracellular compartment and thus impaired platelet activation substantially. Furthermore, the phosphorylation of the Ca(2+) sensor STIM1 was abrogated upon CyPA deficiency, as shown by immunoprecipitation studies. In a mouse model of arterial thrombosis, CyPA-deficient mice were protected against arterial thrombosis, whereas bleeding time was not affected. The results of the present study identified CyPA as an important Ca(2+) regulator in platelets, a critical mechanism for arterial thrombosis.
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- 2012
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30. The serum- and glucocorticoid-inducible kinase 1 (SGK1) influences platelet calcium signaling and function by regulation of Orai1 expression in megakaryocytes.
- Author
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Borst O, Schmidt EM, Münzer P, Schönberger T, Towhid ST, Elvers M, Leibrock C, Schmid E, Eylenstein A, Kuhl D, May AE, Gawaz M, and Lang F
- Subjects
- Animals, Bleeding Time, Blotting, Western, Calcium Channels genetics, Cells, Cultured, Female, Flow Cytometry, Fluorescent Antibody Technique, Humans, Immunoenzyme Techniques, Leukemia, Megakaryoblastic, Acute metabolism, Leukemia, Megakaryoblastic, Acute pathology, Male, Megakaryocytes cytology, Mice, Mice, Knockout, NF-kappa B genetics, NF-kappa B metabolism, ORAI1 Protein, Phosphorylation, Platelet Aggregation, RNA, Messenger genetics, Reverse Transcriptase Polymerase Chain Reaction, Thrombosis etiology, Thrombosis metabolism, Thrombosis pathology, Blood Platelets metabolism, Calcium metabolism, Calcium Channels metabolism, Calcium Signaling, Immediate-Early Proteins physiology, Megakaryocytes metabolism, Protein Serine-Threonine Kinases physiology
- Abstract
Platelets are activated on increase of cytosolic Ca2+ activity ([Ca2+](i)), accomplished by store-operated Ca2+ entry (SOCE) involving the pore-forming ion channel subunit Orai1. Here, we show, for the first time, that the serum- and glucocorticoid-inducible kinase 1 (SGK1) is expressed in platelets and megakaryocytes. SOCE and agonist-induced [Ca2+](i) increase are significantly blunted in platelets from SGK1 knockout mice (sgk1(-/-)). Similarly, Ca2+ -dependent degranulation, integrin α(IIb)β3 activation, phosphatidylserine exposure, aggregation, and in vitro thrombus formation were significantly impaired in sgk1(-/-) platelets, whereas tail bleeding time was not significantly enhanced. Platelet and megakaryocyte Orai1 transcript levels and membrane protein abundance were significantly reduced in sgk1(-/-) mice. In human megakaryoblastic cells (MEG-01), transfection with constitutively active (S422D)SGK1 but not with inactive (K127N)SGK1 significantly enhanced Orai1 expression and SOCE, while effects reversed by the SGK1 inhibitor GSK650394 (1μM). Transfection of MEG-01 cells with (S422D)SGK1 significantly increased phosphorylation of IκB kinase α/β and IκBα resulting in nuclear translocation of NF-κB subunit p65. Treatment of (S422D)SGK1-transfected MEG-01 cells with the IκB kinase inhibitor BMS-345541 (10μM) abolished SGK1-induced increase of Orai1 expression and SOCE. The present observations unravel SGK1 as novel regulator of platelet function, effective at least in part by NF-κB-dependent transcriptional up-regulation of Orai1 in megakaryocytes and increasing platelet SOCE.
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- 2012
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31. SGK1 sensitivity of platelet migration.
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Schmidt EM, Kraemer BF, Borst O, Münzer P, Schönberger T, Schmidt C, Leibrock C, Towhid ST, Seizer P, Kuhl D, Stournaras C, Lindemann S, Gawaz M, and Lang F
- Subjects
- Animals, Cells, Cultured, Chelating Agents pharmacology, Chemokine CXCL12 physiology, Egtazic Acid analogs & derivatives, Egtazic Acid pharmacology, Female, Immediate-Early Proteins genetics, Intestines blood supply, Ischemia metabolism, Ischemia pathology, Male, Mice, Mice, Knockout, Phosphorylation, Protein Processing, Post-Translational, Protein Serine-Threonine Kinases genetics, Signal Transduction, Vinculin metabolism, Wiskott-Aldrich Syndrome Protein metabolism, Blood Platelets physiology, Immediate-Early Proteins metabolism, Protein Serine-Threonine Kinases metabolism, Transendothelial and Transepithelial Migration
- Abstract
Recent observations pointed to the ability of platelets to migrate and thus to invade the inflamed vascular wall. Platelet migration could be stimulated by stromal cell-derived factor-1 (SDF-1), an effect dependent on phosphatidylinositide-3-kinase (PI3K) and paralleled by activation and phosphorylation of Wiskott-Aldrich syndrome protein (WASP). Migration is inhibited by vinculin, which is similarly regulated by phosphorylation. PI3K-sensitive kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored whether SGK1 modifies WASP and vinculin phosphorylation in murine platelets and participates in the regulation of platelet migration. Platelets were isolated from gene-targeted mice lacking SGK1 (sgk1(-/-)) and from their wild type littermates (sgk1(+/+)). Platelet migration stimulated with SDF-1 was significantly less pronounced in sgk1(-/-)platelets than in sgk1(+/+) platelets. Moreover, SDF-1 significantly induced WASP phosphorylation, an effect again reduced in platelets lacking SGK1. Phosphorylation of vinculin was significantly enhanced in sgk1(-/-)platelets and was significantly reduced following treatment of platelets with Ca(2+) chelator BAPTA. Immunohistochemical analysis of in vivo experiments in intestinal vessels after vascular inflammation revealed that transmigration of platelets into inflamed vessel walls was significantly less pronounced in sgk1(-/-)than in sgk1(+/+) mice. In conclusion, SGK1 is a powerful regulator of platelet migration., (Copyright © 2012 S. Karger AG, Basel.)
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- 2012
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32. Ion channels in the regulation of platelet migration.
- Author
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Schmidt EM, Münzer P, Borst O, Kraemer BF, Schmid E, Urban B, Lindemann S, Ruth P, Gawaz M, and Lang F
- Subjects
- Animals, Blood Platelets drug effects, Calcium Channels metabolism, Chloride Channels metabolism, Humans, Intermediate-Conductance Calcium-Activated Potassium Channels metabolism, Mice, Nitrobenzoates pharmacology, ORAI1 Protein, Blood Platelets physiology, Cell Movement, Chemokine CXCL12 metabolism, Ion Channels metabolism
- Abstract
Platelets have been shown to migrate and thus to invade the vascular wall. Platelet migration is stimulated by SDF-1. In other cell types, migration is dependent on Ca(2+) entry via Ca(2+) channels. Ca(2+) influx is sensitive to cell membrane potential which is maintained by K(+) channel activity and/or Cl(-) channel activity. The present study explored the role of ion channels in the regulation of SDF-1 induced migration. Platelets were isolated from human volunteers as well as from gene targeted mice lacking the Ca(2+) activated K(+) channel SK4 (sk4(-/-)) and their wild type littermates (sk4(+/+)). According to confocal microscopy human platelets expressed the Ca(2+) channel Orai1 and the Ca(2+)-activated K(+) channel K(Ca)3.1 (SK4). SDF-1 (100 ng/ml) stimulated migration in human platelets, an effect blunted by Orai1 inhibitors 2-aminoethoxydiphenyl borate 2-APB (10 μM) and SKF-96365 (10 μM), by unspecific K(+) channel inhibitor TEA (30 mM), by SK4 specific K(+) channel blocker clotrimazole (10 μM), but not by Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid NPPB (100 μM). Significant stimulation of migration by SDF-1 was further observed in sk4(+/+) platelets but was virtually absent in sk4(-/-) platelets. In conclusion, platelet migration requires activity of the Ca(2+) channel Orai1 and of the Ca(2+) activated K(+) channel SK4, but not of NPPB-sensitive Cl(-) channels., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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33. Thymoquinone-induced platelet apoptosis.
- Author
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Towhid ST, Schmidt EM, Schmid E, Münzer P, Qadri SM, Borst O, and Lang F
- Subjects
- Blood Platelets cytology, Blood Platelets metabolism, Blotting, Western, Calcium metabolism, Cells, Cultured, Ceramides metabolism, Flow Cytometry, Fluorescence, Humans, Integrin beta3 metabolism, P-Selectin metabolism, Apoptosis drug effects, Benzoquinones pharmacology, Blood Platelets drug effects, Platelet Membrane Glycoprotein IIb metabolism
- Abstract
Thymoquinone (TQ) is a nutrient with anticarcinogenic activity that stimulates suicidal death of tumor cells. Moreover, TQ triggers suicidal death of erythrocytes or eryptosis, an effect at least partially due to increase in cytosolic Ca(2+) activity and ceramide formation. The present experiments explored whether TQ influences apoptosis of blood platelets. Cell membrane scrambling was determined utilizing Annexin V binding to phosphatidylserine exposing platelets, cytosolic Ca(2+) activity utilizing Fluo 3-AM fluorescence, caspase activity utilizing immunofluorescence and Western blotting of active caspase-3 and inactive procaspase-3, mitochondrial potential utilizing DiOC(6) fluorescence and ceramide by FACS analysis of ceramide-binding antibodies. A 30 min exposure to TQ (≥5 µM) was followed by Annexin V binding, paralleled by caspase activation, increase of cytosolic Ca(2+) activity, mitochondrial depolarization, and ceramide formation. P-selectin exposure and integrin α(IIb) β(3) activation did not increase in response to TQ. Nominal absence of extracellular Ca(2+) blunted but did not fully abolish the TQ-induced activation of caspase-3. The effects of TQ on platelets are significantly abolished with phosphoinositide-3 kinase (PI3K) inhibitor wortmannin and G-protein coupled receptor (GPCR) inhibitor pertussis toxin treatment prior to TQ stimulation. In conclusion, TQ triggers suicidal death of blood platelets in a PI3K-dependent manner, possibly through a GPCR family receptor; an effect paralleled by increase of cytosolic Ca(2+) activity, ceramide formation, mitochondrial depolarization, and caspase-3 activation., (Copyright © 2011 Wiley Periodicals, Inc.)
- Published
- 2011
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34. Human Platelets Take up Anti-VEGF Agents.
- Author
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Sobolewska, B., Fehrenbacher, B., Münzer, P., Kalbacher, H., Geue, S., Stellos, Konstantinos, Schaller, M., and Ziemssen, F.
- Subjects
VASCULAR endothelial growth factor antagonists ,PROTEIN kinases ,NEOVASCULARIZATION inhibitors ,STAINS & staining (Microscopy) ,BLOOD platelets ,MICROSCOPY ,BIOLOGICAL transport ,MONOCLONAL antibodies ,MICROFILAMENT proteins ,FLUORESCENT antibody technique ,MEMBRANE proteins ,CYTOPLASM ,CARRIER proteins - Abstract
Purpose. Growing evidence suggests different systemic exposure of anti-vascular endothelial growth factor (anti-VEGF) agents with repeated intravitreal application. Since the penetration of anti-VEGF agents through vascular barrier was reported, the interaction of anti-VEGF with nonresident platelets has become a topic of interest. The purpose of this study was to evaluate, with the help of visualization techniques, whether platelets take up the anti-VEGF agents ranibizumab, aflibercept, and bevacizumab. Methods. The uptake of anti-VEGF agents with or without VEGF treatment was investigated using immunofluorescence and immunogold staining in human platelets. The role of actin filaments and clathrin-coated vesicles in the transport of ranibizumab, aflibercept, and bevacizumab was evaluated by two pharmacologic inhibitors: staurosporine (protein kinase C inhibitor) and cytochalasin D. Results. All three anti-VEGF agents were taken up by platelets and colocalized with VEGF. Ranibizumab and aflibercept were mainly detected in alpha-granules; however, bevacizumab was equally localized in alpha-granules and in platelet vesicles. Both staurosporine and cytochalasin D completely inhibited the uptake of aflibercept into platelets. Both pharmacological inhibitors also decreased the transport of ranibizumab and bevacizumab into platelets. Bevacizumab was significantly more frequently colocalized within clathrin-coated vesicles than ranibizumab and aflibercept. Conclusion. All three anti-VEGF agents are taken up by platelets and internalized in alpha-granules, which may result in a higher local exposure of anti-VEGF after the activation of platelets, potentially contributing to arterial thromboembolic events. Clathrin-coated vesicles seem to be more prominent in the transport of bevacizumab than ranibizumab and aflibercept. Nevertheless, whether the different localization and transport of bevacizumab are truly related to specific differences of receptor-mediated endocytosis has to be revealed by further research. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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